Therapeutic application of cells derived from embryonic stem (ES) or "reprogrammed" stem cells may require, for certain cells types such as hematopoietic stem cells (HSC), expansion of these populations without introducing genetic alterations to induce self-renewal. To address this issue, we propose to test our hypotheses that pathways that regulate normal development can be manipulated to generate expanded populations of cell types that reflect normal, developmental states. As a model for this approach, we focus on the Notch and Wnt pathways with which we have extensive experience. Thus, ES and induced pluripotent stem (IPS) cells will be assessed for development of enhanced numbers of HSC by manipulation of the Notch (project 1) and Wnt (project 2) pathways. As part of each of these projects, global assessment of gene expression and epigenetic state is planned to respectively determine key similarities or differences between in vitro derived hematopoietic cells and isolated normal counterparts. Based on our current generation and clinical application of human CD34+ stem/progenitor cells using the Notch ligand Delta1, we will also test the in vivo biological function of a well characterized in vitro generated stem/progenitor population and assess the significance of potential deviations from "normal" molecular profiles (Project 3). To further understand mechanisms involved in Notch- and Wnt-induced affects on self-renewal and differentiation, we will perform genome-scale, "multiplexed" RNAi screens in expanding populations of ES/IPS and cord blood derived CD34+ cells (project 4). The assembled team represents multidisciplinary expertise at the Fred Hutchinson Cancer Research Center (FHCRC) and the University of Washington (UW) in hematopoietic stem cell biology, Wnt and Notch signaling, epigenetics, evolution of gene expression in development, genome-scale RNAi technology, and requisite experience in ES and IPS. We anticipate providing the expertise, as well as unique reagents for modulating Notch and Wnt signaling, in collaborations with other members of the stem cell consortium evaluating heart and lung as well as hematopoietic progenitors.